Chiral γ-substituted β-hydroxybutyric acid esters are commercially important intermediates in the synthesis of pharmaceuticals. These intermediates may be utilized as optically active intermediates in the synthesis of HMG-CoA reductase inhibitors, such as Atorvastatin, Fluvastatin, and Rosuvastatin. Methods have been described for producing some γ-substituted β-hydroxybutyric acid esters. For example, a method has been reported for producing 4-cyano-3-hydroxybutyric acid from 4-bromo-3-hydroxybutyrate that requires the protection of the hydroxy group with a protecting group prior to reaction with sodium cyanide. Acta Chem. Scand., B37, 341 (1983). Isbell, et al. further report a method for synthesizing (R)-4-cyano-3-hydroxybutyric acid ester by reacting the monohydrate calcium salt of threonine with hydrogen bromide to produce the di-bromo form of threonine, which is then converted to bromohydrin. Carbohydrate Res., 72:301 (1979). The hydroxy group of the bromohydrin is protected prior to reaction with sodium cyanide. Id. Unfortunately, methods requiring protecting and deprotecting steps are not practical to implement commercially.
More recent routes to synthesizing cyanohydrins have been developed that utilize ethyl 4-bromo-3-hydroxybutyrate. These routes require a large number of steps that are relatively costly to carry out commercially.
Description of Ketoreductase
KRED Characterization
Enzymes belonging to the ketoreductase (KRED) or carbonyl reductase class (EC 1.1.1.184) are useful for the synthesis of optically active alcohols from the corresponding prochiral ketone substrate. KREDs typically convert a ketone substrate to the corresponding alcohol product, but may also catalyze the reverse reaction, oxidation of an alcohol substrate to the corresponding ketone/aldehyde product. The reduction of ketones and the oxidation of alcohols by enzymes such as KRED, requires a co-factor, most commonly reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH), and nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) for the oxidation reaction. NADH and NADPH serve as electron donors, while NAD and NADP serve as electron acceptors. It is frequently observed that ketoreductases and alcohol dehydrogenases accept either the phosphorylated or the non-phosphorylated co-factor (in its oxidized and reduced state), but not both.
KRED enzymes can be found in a wide range of bacteria and yeasts (for reviews: Kraus and Waldman, Enzyme catalysis in organic synthesis Vol's 1&2. VCH Weinheim 1995; Faber, K., Biotransformations in organic chemistry, 4th Ed. Springer, Berlin Heidelberg New York. 2000; Hummel and Kula Eur. J. Biochem. 1989 184:1-13; Liese). Several KRED gene and enzyme sequences have been reported, e.g. Candida magnoliae (Genbank Acc. No. JC7338; GI:11360538) Candida parapsilosis (Genbank Acc. No. BAA24528.1; GI:2815409), Sporobolomyces salmonicolor (Genbank Acc. No. AF160799; GI:6539734).
Desired KRED Properties
Metabolism in the living cell ensures the adequate supply of co-factors for reduction reactions by de novo synthesis and regeneration. The use of whole cells for biocatalytic ketone reductions may therefore be advantageous, however, microorganisms typically have multiple ketoreductases which can lead to low product of low enantiomeric excess. For that reason, Wong et al. studied (semi)-purified ketoreductases enzymes and found that higher quality products can be obtained (Wong et al. J. Am. Chem. Soc 1985 107:4028-4031).
In the absence of the cellular machinery during in vitro enzymatic reductions, co-factor regeneration is needed to circumvent the need for stoichiometric amounts of these expensive molecules. The use of enzymes for reduction of ketones therefore requires two enzymes—KRED and a cofactor (NADH or NADPH) regenerating enzyme such as glucose dehydrogenase (GDH), formate dehydrogenase etc. Enzymes are generally considered expensive due to their low activity under process conditions (e.g. Sutherland and Willis, J. Org. Chem. 1998 63:7764; Bustillo et al. Tetrahedron Assym 2002 13:1681), insufficient stability (Shimizu et al. Appl. Environ. Microbiol. 1990 56:2374; Bradshaw et al. J. Org. Chem. 1992 57:1526), and vulnerability to substrate or product inhibition (Kataoka et al. Appl. Microbiol. Biotechnol. 1997 48:699); Kita et al. Appl. Environ. Microbiol. 1999 65: 5207). As mentioned above, co-factors are expensive reagents for industrial processes and may add significant cost to a biological reduction process if their usage is not efficient.
To circumvent many of these perceived economic issues, whole microbial cells have been frequently considered as preferred catalyst for biocatalytic reductions, as they typically contain co-factor and co-factor regenerating enzymes. Asymmetric reduction of 4-chloroacetoacetate esters has been described with bakers yeast (Zhou, J. Am. Chem. Soc. 1983 105:5925-5926; Santaniello, J. Chem. Res. (S) 1984:132-133) and many other microorganisms (U.S. Pat. Nos. 5,559,030; 5,700,670 and 5,891,685). However, reductions using microbial cells are not performed at high substrate concentration are not efficient, suffer from reduced yield due to competing reactions and give low enantiomeric excess (“e.e.”) (U.S. Pat. Nos. 5,413,921; 5,559,030; 5,700,670; 5,891,685; 6,218,156; and 6,448,052).
Introduction of genes encoding KRED and GDH into a fast-growing microorganism such as E. coli has resulted in more active whole cell catalysts for the reduction of ketones. The carbonyl reductase gene from Candida magnoliae and the GDH gene from Bacillus megaterium were cloned in E. coli and allowed for the production of ethyl-4-chloro-3-hydroxybutyrate. To achieve a significant productivity, the NADP co-factor was added to the reaction to provide sufficient activity to the catalyst. At the end of the reaction, the chiral product was extracted and purified by common procedures such as chromatography and distillation. While this procedure is an improvement over processes that use native organisms, significant drawbacks for economic production still persist as NADP continues to be a required additive, and significant process investments are needed to isolate the product in a pure form from the reaction mixture that contains microbial cells.
With these caveats in both enzymatic and whole cell reduction processes in mind, it was an object of the present invention to describe the generation of enzymes, their amino acid sequences and the genes encoding such sequences that facilitate the efficient and economic reduction of ethyl-4-chloro-3-ketobutyrate and other ketones in a clean reaction process. Thus, while microbial reductions typically require cell concentrations of 5 g/L or more, new enzymes are described that catalyze these reactions at enzyme concentrations below 1 g/L, preferably below 0.5 g/L. In addition, the enzymes described, catalyze the complete conversion of at least 100 g/L substrate in less than 20 hrs and require only small amounts of co-factor.
The above referenced patents and publications and all other patents and publications cited throughout this specification are expressly incorporated by reference herein in their entirety.